BACKGROUND OF THE INVENTION
[0001] This invention relates to luminescent reagents having utility for monitoring specific
binding reactions and, more particularly, to a luminol preparation having enhanced
light output and stability.
[0002] Specific binding assays provide an economical means for detecting and measuring an
analyte present in low concentrations in a sample. Specific binding assays are based
upon the interaction of two bindable substances, one the analyte and the other a specific
binding partner, which specifically recognize each other. Examples of specific binding
partners whose interaction can serve as the basis for a specific binding assay include
antigens-antibodies, biotin-avidin, nucleic acid probes, enzymes-substrates, enzymes-inhibitors,
enzymes-cofactors, chelators-chelates, and cell surface receptor pairs. Assays involving
other specifically bindable substances are also known and within the scope of the
present invention. Specific binding assays have shown great utility in determining
various analytes in biological, medical, environmental, agricultural and industrial
applications.
[0003] A variety of assays using the principles of the specific binding approach are known,
and several have become important diagnostic tools. In one such type of specific binding
assay, the immunoassay, the analyte is an antibody, antigen, or hapten, and is made
to react with another member of this group. While the background discussion will focus
on such immunoassays, this focus is made for clarity of presentation, and is not to
be interpreted as limiting of the invention, which is broadly applicable to luminescently
labelled specific binding assays.
[0004] A variety of labelling reactions have been proposed for use in specific binding assays,
including radioactive, enzymatic, chromogenic and luminogenic procedures. In a radioactive
labelling procedure, the component conjugated with the specific binding partner is
an atom or molecule which emits radioactivity. Chromogenic and luminogenic labelling
reactions are chemically more complex, in that several reactants may be involved.
The chromophore or lumiphore may itself be the label in the reaction, or a catalyst,
typically an enzyme, may be used as the label. When the catalyst is used as the label,
it will react with catalytic substrates which in turn produce color or luminescence.
The remaining components of the reaction, that is, those not conjugated to the binding
partner, are supplied in a chromogenic or luminogenic reagent medium, so that the
uniting of the labelled conjugate and the reagent medium results in the desired color
change or light emission, respectively.
[0005] Luminescent labels are attractive alternatives for use in specific binding assays
for a variety of reasons. Luminescence is broadly defined a= the production of visible
light by atoms that have been excited by the energy produced in a chemical reaction,
usually without an associated production of heat. Chemical energy excites electrons
in the light-emitting molecules to higher energy states, from which electrons eventually
fall to lower energy states with the emission of quanta of energy in the form of visible
light. Luminescence is observed in several synthetic chemical compounds and also in
naturally occurring biological compounds such as found in fireflies and certain species
of fish.
[0006] One of the most important families of chemiluminescent molecules are the phthalyl-
hydrazides. The most familiar member of this family is luminol, or 5-amino-2,3-dihydro-1,4-phthalazine-
dione, which has a gross chemical composition of C
aH
7N
30
2 and a double ring structure with a melting point of about 320
.C. Luminol is commercially available from several suppliers and is well characterized.
Certain luminol analogs are also chemiluminescent, such as those wherein the position
of the amino group is shifted (e.g., isoluminol, the amino group being at the 6 position),
or is replaced by other substituents, as well as annelated derivatives and those with
substitution in the nonheterocyclic ring. Some luminol analogs produce light more
efficiently than does luminol itself, while others have lower efficiency. (As used
herein, the term "luminol" encompasses such related species.)
[0007] Generally, luminol produces light in an oxidizing reaction, wherein the luminol combines
with oxygen or an oxidizer to produce a reaction product and photons at a wavelength
of about 425-450 nanometers (nm). The precise reaction formula and the quantum efficiency
of light production (that is, the ratio of luminescing molecules to total molecules
of the luminescent species) depend upon the medium in which the luminol resides, temperature
and other reaction conditions. Typical oxidizers used in conjunction with luminol
include oxygen, hydrogen peroxide, hypochloride, iodine, and permanganate.
[0008] The oxidation of luminol with the associated production of light occurs rather slowly
at ambient temperatures, unless the reaction is catalyzed. A variety of different
substances can catalyze the reaction, including organic enzymes (for example, horseradish
peroxidase), other organic molecules such as microperoxidase and heme, positive metallic
ions such as the cupric ion, and negative ions such as the ferricyanate ion.
[0009] Luminescent molecules would appear to be highly desirable as tags in specific binding
assays because of their stability, sensitivity, the potential ease of detecting their
emitted visible light and their lack of toxicity. Commercial luminol, however, has
proven to be unsuitable for such purposes. There exists a need for specific improvements
in the light emission characteristics of the reaction for use with such assays. Heretofore,
commercial luminol has not shown sufficient activity to be useful to measure analytes
at low concentrations in specific binding assays. The light emission intensity of
the luminol reaction may be sufficient where high concentrations of catalyst are employed
and where highly sophisticated and sensitive photometers are available, but the luminescent
intensity has not been sufficient with low concentrations of catalyst and where other
detection media such as photographic film or less sensitive photometers are used.
[0010] While the luminol reaction therefore offers important potential benefits in the measurement
of the presence and amount of a reaction component, for many potential applications
the intensity of the emitted light is too low. Further, the light emitted from commercial
luminol exhibits an early flash of light within the first few seconds of the initiation
of the reaction, followed by a progressive and rapid decrease in light emission over
time. The integrated light intensity during any fixed period of time is therefore
likely to be different from that measured over any other equal period of time. This
variability may result in irreproducibility between tests. Desirably, there would
be some period of time during which the light emission from the luminol reaction is
relatively constant, so that the measurement of integrated light intensity could begin
at different times after initiation of the reaction but within the period of constant
light output, without variability of the results. This would eliminate the requirement
that the reagents be added to a solution fixed in front of the luminescence detector
which puts severe constraints on the light measuring system.
[0011] Inhibitors for the catalysts used in luminescent reactions have been reported (Theorell,
The Enzymes, Vol. II, Part I, p. 397, Academic Press (1951)). However, the need to
rigorously remove these inhibitors from luminogenic substrates has heretofore not
been appreciated. We have discovered that rigorous removal of inhibitors from the
luminescent substrates produces substantial improvement in the resulting sensitivity
and reproducibility in specific binding assays. This is especially important when
measuring low concentrations of analyte which necessitates low concentrations of catalyst.
[0012] There therefore exists a need for an improved luminescent substrate for use in specific
binding assays and other applications. The improved luminescent substrate should exhibit
an increased unit light intensity output per molecule of reacting luminescent substrate
to allow its use as a tag in specific binding assays such as immunoassays where the
concentration of analyte is low. The improved luminescent substrate should exhibit
a period of substantially constant light output that would allow repetition of test
procedures and eliminate many constraints on the user and the light detection system.
The present invention fulfills these needs, and further provides related advantages.
SUMMARY OF THE INVENTION
[0013] The present invention provides an improved luminescent substrate preparation exemplified
by a luminol preparation, its method of preparation and its use in luminescent assays.
The luminol preparation exhibits an increased light output of about at least a factor
of ten, as compared with typical samples of commercially available luminol. Further,
the luminol preparation also exhibits a more uniform light output as a function of
time than untreated luminol when used in a specific binding assay.
[0014] In accordance with the present invention, a method for making the luminol preparation
from untreated commercial luminol is provided which comprises the steps of furnishing
commercial luminol; dissolving the luminol in an alkaline solution; heating the solution;
and separating crystals of the luminol preparation from the heated solution. The steps
of dissolving, heating and separating are preferably repeated sequentially, in each
sequence using as the luminol starting material the crystals prepared in the prior
sequence. In a preferred embodiment, it is found that four repetitions provide a significantly
enhanced luminol preparation, .and that fewer or greater repetitions may be justified,
given the constraints required for the particular assay.
[0015] It is believed that the enhanced characteristics of the luminol preparation result
from a reduction in the level of substances which inhibit chemiluminescent reactions,
specifically by inhibiting the activity of catalysts, e.g., peroxidase, used in monitoring
specific binding assays. For example, hydrazine and sulfide ions inhibit the peroxidase
reaction, and that one or both such inhibitors as well as others may be present in
commercially available luminol as a result of the synthesis procedure used in the
preparation of luminol.
[0016] In accordance with another aspect of the invention, a luminol preparation is provided
which has a concentration of inhibitors of less than 100 parts per million. The luminol
preparation exhibits a unit light output per molecule of reacting luminol approximately
ten times greater than that of commercially prepared, untreated luminol. The increased
unit light output allows more sensitive measurements of chemical reactions in qualitative
and quantitative tests. Moreover, whereas the luminescence emitted by commercially
available, untreated luminol shows a substantial decrease in light emission over time
after initiation of the reaction, the luminol preparation reported herein shows a
dramatic improvement in that the luminescence emission over time does not substantially
decrease. It will be appreciated that these enhancements to luminol constitute an
important improvement in the art of luminescent reactions, particularly where such
reactions are used to measure low concentrations of analyte in specific binding assays.
[0017] In accordance with a further aspect of the invention, the luminol preparation is
used to monitor luminescent specific binding assays. Until now, the most commonly
used tags for monitoring specific bind- ir.g assays have been various radioactive
ions, such as
125I. Because of the safety problems involved in handling and disposing of radioactive
materials, it is highly desirable to utilize alternative tagging materials. Luminescent
materials, which predictably emit light under certain conditions, are an obvious choice
and have been used for certain applications. commercial luminol has until now been
inappropriate for use in particular types of assays, such as specific binding assays,
where the concentration of analytes to be measured is low, because of its low level
of light emission per molecule. The luminol preparation of the present invention,
however, possesses enhanced emission characteristics which render it useful in such
specific binding assays.
[0018] In a preferred embodiment, the luminol preparation is used to monitor the presence
of antibodies specific for various allergens (IgE) in the serum of patients. Allergens
are immobilized on a solid support, such as cotton threads, and contacted with a sample
of human serum to allow binding between the allergens and corresponding antibodies.
The solid support is then contacted with a solution containing anti-IgE antibodies
which have been labelled with peroxidase. After incubation to allow binding between
any IgE present and the anti-IgE antibody, the threads are contacted with a solution
containing luminol and peroxide. In the presence of the catalyst peroxidase, the luminol
emits light, permitting localization of the tagged anti-human antibody and, ergo,
the IgE.
[0019] It will be appreciated that these enhancements of luminol constitute an important
improvement in the art of luminescent reactions. Other features and advantages of
the present invention will become apparent from the following more detailed description
which illustrates, by way of example, the principles of the invention.
DETAILED DESCRIPTION
OF THE PREFERRED EMBODIMENT
[0020] The synthesis of luminol is reported in references listed in entry number 5413 of
the Merck Index, which is herein incorporated by reference, and need not be set forth
in detail herein. However, common to the synthesis procedures is the use of hydrazine
and sulfur containing compounds. For example, in Huntress, J. Am. Chem. Soc. 56:241
(1934), hydrazine is used to synthesize 3-nitro- phthalhydrazide, and this compound
is then dissolved in ammonium sulfide during the preparation of luminol. In the procedure
set forth in Redemann, Ora. Syn. 29:78,8 (1949), hydrazine sulfate is used to prepare
a mixture of sodium sulfate and 5-nitro-2,3,dihydro-1,4-phthalazinedione, and this
compound is converted to luminol, in part through the use of sodium hydrosulfite dihydrate.
Thus, in both synthesis procedures, hydrazine and sulfur containing compounds are
intentionally utilized in synthesizing the luminol. As noted in the Redemann discussion,
the intermediate product may contain small amounts of inorganic salts, which may be
carried through the final luminol product.
[0021] Luminol is available commercially as crystals or as a fine yellowish powder from
several sources, including: Sigma Chemical Company, St. Louis, MO; Aldrich Chemical
Company, Inc., Milwaukee, WI; Mallinckrodt, St. Louis, MO; Fisher Scientific Company,
Pittsburgh, PA. The sources do not disclose tne synthesis procedure, and it is therefore
not possible to state with certainty the procedure used. The procedure may be one
of those disclosed publicly, or yet other synthesis procedures may be followed. One
important feature of the present invention is the ability to improve a variety of
commercially available, untreated luminols, without any knowledge of the procedure
used in synthesizing the luminol.
[0022] Regardless of the synthesis procedures used and the reasons underlying the effect,
the user of commercially purchased, untreated luminol must contend with a low level
of light output from the material. While these phenomena may be acceptable to some
users of luminol, in sensitive quantitative analysis procedures the variability and
low light output can pose significant problems. These problems could conceivably be
overcome through the use of specialized measurement apparatus or, as in the present
case, by improving luminol so as to avoid the problems. As used herein, the term "luminol
preparation" refers to a material which is prepared from untreated luminol, typically
commercially available luminol, and exhibits intensified unit light output.
[0023] In accordance with the invention, a luminol preparation is prepared by furnishing
commercially prepared, untreated luminol, dissolving the luminol in an alkali solution
to form a crude solution, boiling the crude solution to form a heated luminol solution,
and separating activated luminol crystals from the heated luminol solution. This procedure
is repeated as necessary to achieve a luminol preparation of sufficient quality to
accommodate the constraints dictated by the specific binding assay in which it is
used. The luminol preparation can be used as a tag in standard specific binding assay
formats, such as those disclosed in Maggio, Enzyme immunoassay, CRC Press (1980),
which is incorporated herein by reference.
EXAMPLE 1
PREPARATION OF LUMINOL
TO REMOVE CATALYTIC INHIBITORS
[0024] In a preferred activation procedure for preparing approximately 18-28 grams of activated
luminol from 100 grams of untreated, commercially prepared luminol (Mallinckrodt,
St. Louis, MO), 22.
6 grams of sodium hydroxide is dissolved in 188 milliliters of distilled water. One
hundred grams of untreated luminol is added to this sodium hydroxide solution and
stirred until dissolved, to achieve an alkaline pH, preferably between 11 and 14 and
most preferably 12-13. A second volume of 188 milliliters of distilled water is then
added to the mixture. The mixture is heated in a glass container to the boiling point,
about 100°C., for a period of time of from about 60 to 120 minutes. The boiled solution
is cooled to the temperature range of from about 50 to about 80
.C., and poured through a 5 micron membrane filter. The filtered solution is cooled
to a temperature of from about 0°C. to about -50°C., to initiate the growth of crystals
in the container. The crystals are allowed to grow for at least 1 hour, and preferably
8 hours, after crystallization first begins, and then filtered to collect the crystals.
The crystals are washed with cold anhydrous alcohol and dried. They are then dissolved
in water and the solution acidified with glacial acetic acid to pH 5-6. At this point,
the solution becomes pasty and must be stirred well to insure a uniform distribution
and pH. The luminol paste is filtered and washed with cold water (about 4
.C.) until acetic acid has been washed away or the filtrate returns to pH 7.0.
[0025] The unit light intensity output of the luminol preparation is observed to be substantially
greater than that of the untreated luminol. The light intensity output may be further
improved by repeating the treatment procedure previously described.
[0026] As is apparent, the treatment sequence may be repeated as many times as desired,
with the ultimate end point being determined by a trade-off between improved light
output properties, the reduced yield of activated luminol with each succeeding sequence
and the particular requirements of the specific binding assay being measured. A total
of four treatment sequences is presently preferred, based upon the improved properties
of the thus prepared luminol, considerations of the yield of the process and the particulars
of the specific binding assay.
EXAMPLE 2
LIGHT EMISSION OF LUMINOL PREPARATION
[0027] A standardized test procedure for measuring luminol light output has been established.
To 0.5 ml of 50mM borate buffer, pH 9:4, are added 0.5 ml of 40mM luminol in 45mM
NaOH, pH 11.0; 0.5 ml of 4mM hydrogen peroxide in 0.01M phosphate buffered saline
(PBS), pH 7.0; 0.4 ml of deionized water;and 0.1 ml of 200 mU/ml horseradish peroxidase
(HRP) to yield a final 2.0 ml volume containing: lOmM luminol; 1 mM H
20
2; 12.5mM borate buffer, pH 9.4; and 20 mU HRP in an aqueous solution having a final
pH of 9.4. The enzyme HRP is added last, the solution mixed and relative intensity
readings taken on an Ames Fluorocolorimeter (Miles Labs, Inc., Elkhart, IN) at various
time points from 1 minute to 2 hours.
EXAMPLE 3
EFFECT OF INHIBITORS ON LUMINESCENT REACTION
[0029] The chemical synthesis of luminol and other luminescent substrates use chemicals
which can inhibit the catalysts used in luminescent reactions. Two of these inhibitory
chemicals known to be used in luminol synthesis were added back to a luminescent reaction
using the luminol prepared in Example 1 above. The reaction conditions were as detailed
in the example using the light emission at 30 minutes as the indicator for the reaction,
except that varying concentrations of ammonium sulfide, hydrazine sulfate and ammonium
sulfate were added to the reactions prior to the addition of the catalyst. The results
were as indicated in Table III:
[0030] As shown, the presence of sulfide and hydrazine ions drastically inhibits the luminescence
yield in the reaction. The presence of their counter ions, as demonstrated by the
ammonium sulfate reactions, had no effect.
[0031] This experiment demonstrates that the luminol must be at least 99.99% free of inhibiting
substances in order to obtain the sensitivity required in specific binding assays
measuring very low concentrations of analytes.
EXAMPLE 4
USE OF LUMINOL PREPARATION
[0032] The luminol preparation may be successfully used to detect the presence of a specific
binding reaction as, for example, in a standard immunoassay format. In a preferred
embodiment, allergens to which a patient is suspected of having a hypersensitivity
are immobilized on a solid support, such as cotton threads. A series of such threads,
each coated with a different allergen can then be mounted together in a spaced relationship
for simultaneous exposure to a serum sample of the patient. The serum is then removed
and the threads washed. The threads are then exposed to a solution containing anti-human
IgE antibodies which have been labelled with a component of the luminol reaction,
preferably the catalyst horseradish peroxidase, and any excess washed off. The threads
are then exposed to a solution containing the treated luminol preparation and peroxide.
Any horseradish peroxidase conjugated anti-human IgE linked to the threads will catalyze
a localized chemiluminescent reaction resulting in light emission from the treated
luminol. The relative amount of anti-human IgE linked to an individual thread can
be determined by monitoring the light emitted adjacent the thread. Because of the
enhanced emission and stability of the treated luminol, the amount of light output
can be monitored by exposing photographic film, such as Polaroid Type 57 to the threads
in solution. The degree of exposure of the film indicates the amount of anti-human
IgE and therefore the amount of IgE complementary to each allergen. The demonstration
of the use of the activated luminol preparation in a specific binding assay is found
in Brown, Clin. Chem., in press (1985).
[0033] While the following explanation is not intended as binding, it is believed that the
treatment sequence described above improves the light-emitting efficiency of.luminol
by reducing the level of trace quantities of substances which inhibit the luminol
light-producing reaction. Specifically, inhibition studies presented herein show that
hydrazine and sulfide ions, when present in amounts greater than about 100 ppm in
the luminol, can inhibit the production of light in the catalyzed luminol oxidation
reaction. Since both hydrazine and sulfur-containing compounds are utilized in the
synthesis of luminol, it is quite possible that trace quantities of these materials
might remain in the crystals after synthesis. Because of the relatively small amounts
of impurities that can reduce light output and the possibility of minor deviations
from synthesis procedures, it is also quite possible that the quantities of these
inhibitors might vary among batches, suppliers, and synthesis techniques. Removal
of the inhibitors thus allows the catalyzed oxidation of luminol to proceed with increased
unit light intensity.
[0034] As will now be appreciated, the activated luminol preparation and the process of
making same of the present invention provide an approach for achieving increased unit
light intensity in the catalyzed oxidation of luminol, and also increased uniformity
of light output with time. The process sequence for treating the luminol is relatively
simple and does not involve complex technology or dangerous chemicals. The light intensity
of the catalyzed luminol oxidation reaction is multiplied by a factor of at least
about ten, thus increasing its sensitivity, decreasing the time necessary for measurements
of the light intensity, and allowing experiments or studies to be repeated at constant
light output if an error is made in the first measurement. Those skilled in the art
will recognize that variations of the preparation procedures described herein may
be made within the spirit and scope of the invention. In particular, the process for
activating the luminol may be varied within the broad scope of the disclosure, yet
achieve substantially the same results in activating the luminol for improved uniformity
and increased light output intensity. Accordingly, the invention is not to be limited
except as by the appended claims. The features disclosed in the foregoing description,
in the claims and/or in the accompanying drawings may, both separately and in any
combination thereof, be material for realising the invention in diverse forms thereof.
1. A composition of matter comprising a luminescent substrate having catalytic inhibitors
in a concentration of less than about 100 ppm.
2. The composition of matter as recited in claim 1, wherein the luminescent substrate
is an phthalylhydrazide derivative.
3. The composition of matter as recited in claim 2, wherein the phthalylhydrazide
derivative is luminol.
4. The composition of matter as recited in claim 1, wherein the catalytic inhibitors
are selected from the group consisting of sulfide and hydrazine ions.
5. A process for preparing luminescent substrates comprising the steps of multiple
recrystallizations to remove inhibitors of the luminescent reaction.
6. A process for preparing luminol, comprising the steps of:
a. dissolving untreated luminol in an alkaline solution:
b. heating the solution; and
c. separating from said solution luminol having catalytic inhibitors in a concentration
of less than about 100 ppm.
7. The process of claim 6, wherein said steps of dissolving, heating and separating
are sequentially repeated, beginning with untreated luminol and using the treated
luminol from the prior sequence as the starting material in each succeeding sequence.
8. The process of claim 7, wherein the sequence of said steps of dissolving, heating
and separating is repeated four times.
9. The process of claim 6, wherein said alkaline solution is sodium hydroxide.
10. The process of claim 9, wherein the concentration of said sodium hydroxide is
about three molar.
11. The process of claim 6, wherein said heating step comprises boiling for a time
of from about 60 to about 120 minutes.
12. The process of claim 6, wherein said step of separating includes the substeps
of:
a. filtering the heated solution;
b. crystallizing the filtered solution by cooling;
c. collecting crystals obtained from said step of crystallizing;
d. treating said crystals with acid.
13. A luminescent substrate prepared by the process of claim 5.
14. Luminol prepared by the process of claim 6.
15. A process for preparing luminol, comprising reducing the concentration of hydrazine
and sulfide ions to less than 100 ppm each.
16. A luminol preparation, comprising crystals of 5-amino-2,3-dihydro-1,4-phthalazinediona,
said crystals having a concentration of catalytic inhibitors of less than about 100
ppm.
17. 'A process for conducting a specific binding assay for detecting an analyte in
a test solution, said specific binding assay being monitored by a luminescent reaction,
comprising:
a. preparing luminol having a concentration of catalytic inhibitors of less than about
100 ppm;
b. introducing to the analyte a specific binding partner thereof, said specific binding
partner being conjugated to a catalyst of the luminescent reaction;
c. exposing said specific binding partner bound to said catalyst to said luminol in
the presence of the other components of the luminescent reaction; and
d. ,.-monitoring light emitted from said luminescent reaction so as to determine whether
said specific binding partner is bound to said analyte.
18. The process of claim 17, wherein said analyte is selected from the group consisting
of antigens, haptens and antibodies.
19. The process of claim 17, wherein said analyte is IgE or anti-IgE.
20. A specific binding reaction assay kit for determining the presence of analytes
in a test liquid, comprising
a. a test chamber having means for introducing liquid into said test chamber, said
test chamber having located therein at least one test surface having fixed thereto
a binding partner for an analyte suspected of being present in the test liquid;
b. a first liquid component having therein a species specifically reactive with analytes
in the test fluid, said species being conjugated with a catalyst of a luminescent
reaction; and
c. a second liquid component comprising an enhanced luminol preparation having catalytic
inhibitors in a concentration of less than about 100 ppm.